Human preprogastrin, a peptide of 101 amino acids, is the primary translation product of the gastrin gene and has the following structure:
(SEQ ID NO: 1)MQRLCVYVLI FALALAAFSE ASWKPRSQQP DAPLGTGANR DLELPWLEQQ GPASHHRRQL GPQGPPHLVA DPSKKQGPWL EEEEEAYGWM DFGRRSAEDE N.
Progastrin is formed by cleavage of the first 21 amino acids (constituting the signal peptide) from preprogastrin. The 80 amino acid long chain of progastrin is further processed by cleavage and modifying enzymes to several biologically active gastrin hormone forms, including gastrin-17 (G17), gastrin-34 (G34), glycine extended gastrin-17 (G17-Gly), and glycine extended gastrin-34 (G34-Gly).
Mature G17 is modified at both amino- and carboxy-terminal residues: the N-terminal glutamine is cyclized to form pyroglutamic acid (pGlu) and the free carboxyl group of the C-terminal phenylalanine residue is amidated by the enzyme, peptidyl α-amidating mono-oxygenase (PAM) to form a C-terminal Phe-NH2. Mature G34 is identically amidated at its C-terminal end to form a C-terminal Phe-NH2 (See Dockray et al., Ann. Rev. Physiol., 63:119-139 (2001)).
Mature G17, the predominant form of “little” gastrin in humans, has the amino acid sequence: pEGPWLEEEEEAYGWMDF-NH2 (SEQ ID NO: 2). G17-Gly is an incompletely processed form of gastrin found as a minor component of “little” gastrin in healthy human subjects and has the amino acid sequence: pEGPWLEEEEEAYGWMDFG (SEQ ID NO: 3).
Gastrin-34, the predominant form of “big” gastrin in humans, has the amino acid sequence: pELGPQGPPHLVADPSKKQGPWLEEEEEAYGWMDF-NH2 (SEQ ID NO: 4). Glycine-extended gastrin 34 (G34-Gly) has a C-terminal glycine residue, and has the amino acid sequence:
(SEQ ID NO: 5)pELGPQGPPHLVADPSKKQGPWLEEEEEA YGWMDFG.
Gastrin is secreted by the pyloric antral-G cells of the stomach in response to gastrin-releasing peptide (GRP), and is suppressed by gastric acid and the paracrine action of several peptide hormones, most notably, somatostatin. It has long been recognized that gastrin peptides function to stimulate acid secretion in the stomach of healthy individuals, however, it has only recently been shown that these peptides also control proliferation, differentiation and maturation of different cell types in the gastrointestinal (GI) system.
Progastrin is normally fully processed to gastrin hormone forms. When produced in excess, progastrin is at least partly processed to one or more forms of gastrin hormone that act on the gastrointestinal system and may potentiate the formation of gastrin-promoted tumors. In some cases the progastrin is circulated in the blood and can be detected in the urine of patients suffering from progastrin-promoted diseases or conditions.
In addition to their local activity in the GI system, G17 and, to a lesser extent, G17-Gly are released into the bloodstream and have been found to increase in the serum of patients afflicted with gastrointestinal disorders and diseases, such as gastric cancer, colorectal cancer, and pancreatic cancer. These gastrin species have more recently also been found to be associated with other diseases not associated with the gastrointestinal tract, including small cell lung cancer (SCLC) and liver metastasized tumors. See for example Joshi et al., “Gastrin and Colon Cancer: a unifying hypothesis” Digestive Diseases, 14:334-344 (1996); and Smith, A. M., and Watson, S. A., “Gastrin and Colorectal Cancer” Alimentary Pharmacology and Therapeutics, 14(10):1231-1247 (2000)).
Antibodies are key reagents in numerous assay techniques used in medical, veterinary and other immunodetection fields. Such tests include many routinely used immunoassay techniques, such as for example, enzyme-linked immunosorbant assays (ELISA), radioimmunoassays (RIA), immunohistochemistry (IHC), and immunofluorescence (IF) assays.
Anti-gastrin polyclonal antibodies have been shown to be effective in inhibiting gastrin activity (Jaffe et al., “Inhibition of gastrin activity by incubation with antibodies to the C-terminal tetrapeptide of gastrin” Surgery, 65(4):633-639 (1969)); and non-human anti-gastrin polyclonal antibodies have been applied to therapy in a patient suffering from Zollinger-Ellison syndrome, a pathological condition in which excessive gastrin is produced without stimulation by feeding. See Hughes et al., “Therapy with Gastrin Antibody in the Zollinger-Ellison Syndrome” Digestive Diseases, 21(3):201-204 (1976). However, these rabbit anti-gastrin antibodies had “at best a short term effect in this patient.” (Hughes et al. at page 204). U.S. Pat. Nos. 5,886,128 and 5,785,970 disclose methods of treatment of ulcers or tumors whose growth is dependent on or stimulated by gastrin hormones by immunizing with gastrin hormone peptide conjugates.
Until now, MAbs capable of sensitively detecting, and accurately distinguishing progastrin from the processed forms of gastrin hormone have not been available. Furthermore, until the present invention, it was not possible to accurately measure the amount progastrin in a sample, such as for instance a sample of biological fluid. The MAbs of the invention can be used in assays for clinical testing to precisely define the biology of progastrin in normal and disease states. The invention also provides MAb compositions for pharmaceutical use and methods for the prevention and treatment of progastrin-promoted diseases and conditions.